Septicemia (or sepsis) is a leading cause of morbidity and mortality among hospitalized patients. Ziegler et al., New Eng. J. Med., 324, 429-35 (1991). There are approximately 400,000 cases each year in the United States, and the incidence continues to increase. Id. Gram-negative bacteremia occurs in about 30% of patients with septicemia. Id. Despite the use of antibiotics and intensive care, the mortality among patients with sepsis and gram-negative bacteremia remains as high as 20-60% depending on the specific population. Id.
Bacteremia and septic shock are associated with the release of endotoxins into the circulation. Id. Endotoxins are the lipopolysaccharide components of the outer membranes of gram-negative bacteria that trigger many of the adverse systemic reactions and serious sequelae in patients with sepsis and gram-negative bacteremia. Id. Endotoxins consist of a polysaccharide portion and a lipid portion (lipid A). The polysaccharide portion contains species-specific (O chain) and group-specific (core) components, i.e. the composition of the core is common to one group of bacteria, whereas the O chain varies from species to species within a genus. For instance, more than 1000 species-specific serotypes of Salmonella have been characterized for one core component. The lipid A moiety is common to all endotoxins and is primarily responsible for the toxicity of endotoxins.
Human polyclonal antiserum specific for endotoxin has been shown to reduce mortality in patients with gram-negative bacteremia and to protect high-risk surgical patients from septic shock. Id. This antiserum was developed by immunizing volunteers with heat-inactivated cells of the J5 mutant of Escherichia coli 0111:B4 which induced an immune response to the core region of endotoxin. The article states that this region is shared among gram-negative bacterial species and contains lipid A.
A human monoclonal antibody (HA-1A) specific for the lipid A domain of endotoxin has been successfully tested in animal models of gram-negative bacteremia and endotoxemia. Ziegler et al., New Eng. J. Med., 324, 430 (1991); Teng et al., Proc. Natl. Acad. Sci. USA, 82, 1790-94 (1985); Ziegler et al., Clin. Res., 35, 619A (1987). HA-1A has been shown to bind specifically to many endotoxins and to a broad range of clinical isolates of gram-negative bacteremia. Ziegler et al., New Eng. J. Med., 324, 430 (1991).
The results of a large double-blind, placebo-controlled trial of the HA-1A monoclonal antibody were reported in Ziegler et al., New Eng. J. Med., 324, 429-435 (1991). Of 200 patients with gram-negative bacteremia proved by blood culture, 105 patients received the HA-1A antibody and 92 patients received placebo. Among the patients receiving the antibody, the mortality rate was 30% over a 28 day period; among patients receiving placebo, the mortality rate was 49%. No benefit from treatment with HA-1A was demonstrated in an additional 343 patients with sepsis who did not prove to have gram-negative bacteremia.
NPC 15669 (N-[9H-(2,7-dimethylfluoren-9-ylmethoxy)carbonyl]-L-leucine) is an anti-inflammatory agent. Noronha-Blob et al., Eur. J. Pharmacol., 199, 387-88 (1991) recently reported that NPC 15669 could reverse endotoxin-mediated leukopenia and reduce mortality from endotoxic shock in mice. Mice pretreated with NPC 15669 two hours prior to a lethal dose of endotoxin "were afforded complete protection (100% survival)." Id. at 387. The authors suggest that NPC 15669 may be of significant therapeutic value in the treatment of septic shock.
Others have tried to control or avoid the effects of endotoxins by using endotoxin-binding adsorbents to remove endotoxins from blood, plasma, and other fluids. See Mitzner et al., Artficial Organ, 15, 338 (1991); Nagaki et al., Artificial Organ, 15, 338 (1991); Nanbu et al., Artificial Organ, 15, 290 (1991). Such endotoxin-binding adsorbents are particularly important in dialysis procedures.
C-reactive protein (CRP) was first described by Tillett and Francis [J. Exp. Med., 52,561-71 (1930)] who observed that sera from acutely ill patients precipitated with fraction C of the cell wall of Streptococcus pneumoniae. Others subsequently identified the reactive serum factor as protein, hence the designation "C-reactive protein."
In addition to binding to pneumococcal C-polysaccharide, CRP binds to: 1) phosphate monoesters, including particularly phosphorylcholine; 2) other cell wall polysaccharides containing phosphorylcholine; 3) phosphatidyl choline (lecithin); 4) fibronectin; 5) chromatin; 6) histones; and 7) the 70 kDa polypeptide of the U1 small nuclear ribonucleoprotein. Kilpatrick and Volanakis, Immunol. Res., 10, 43-53 (1991). Several laboratories have also reported the binding of CRP to galactose-containing polysaccharides. Id. However, one laboratory has reported that CRP binds to trace phosphate groups that are minor constituents of one particular galactan, making it is unclear whether CRP binding to other galactans is also directed to phosphate residues or to carbohydrate determinants. Id.
Xia et al., FASEB J., 5, A1628 (1991) describes experiments designed to explore the role of CRP in endotoxin shock. A chimeric gene coding for rabbit CRP under the control of an inducible promoter (inducible in response to demand for gluconeogenesis) was introduced into mice. In contrast to most other vertebrates, mice synthesize only trace amounts of endogenous CRP, even during an acute phase response. When the chimeric gene was introduced into mice, rabbit CRP was expressed in response to demand for gluconeogenesis. Further, it was found that 75% of mice expressing high levels of rabbit CRP following induction of gluconeogenesis survived treatment with 350-400 .mu.g of endotoxin, as compare to 27% survival for animals in which rabbit CRP synthesis had been suppressed by inhibiting gluconeogenesis. The authors speculate that CRP may play a role in natural defense against endotoxin shock, although CRP is not known to bind endotoxin.
Mold et al., Infection and Immunity, 38, 392-395 (1982) reports that CRP binding can lead to complement activation and, in the presence of complement, enhancement of opsonization of C-polysaccharide-sensitized erythrocytes and type 27 S. pneumoniae. The article further reports that injection of CRP increased survival in mice challenged with type 3 or type 4 S. pneumoniae. Finally, the authors describe test results from which they conclude that CRP binds to a small group of potentially pathogenic gram-positive bacteria (S. pneumoniae, Streptococcus viridans, and one isolate of Staphylococcus aureus), but does not bind to gram-negative bacteria or to other gram-positive bacteria. They, therefore, postulate that the ability of CRP to enhance opsonization and contribute to host defense may be specific for infection with S. pneumoniae.
Similarly, Mold et al., Ann. N.Y. Acad. Sci., 389, 251-62 (1982) reports that CRP can act as an opsonin in the presence of complement. However, the article teaches that CRP does not bind to gram-negative bacteria and binds to only some gram-positive organisms. For those gram-positive bacteria to which CRP binds, the effectiveness of CRP as an opsonin varied depending on the species. Finally, the article reports that CRP protected mice from S. pneumoniae infection.
Nakayama et al., Clin. Exp. Immunol., 54, 319-26 (1983) also teaches that CRP protects against lethal infection with type 3 or type 4 S. pneumoniae. The article further teaches that CRP did not protect against a similar dose of Salmonella typhimurium LT2.
Horowitz et al., J. Immunol., 138, 2598-2603 (1987) describes the effects of CRP in mice with a X-linked immunodeficiency ("xid mice") which prevents the mice from making antibodies to polysaccharide antigens. In these mice, CRP provided protection against infection with type 3 S. pneumonia and acted by clearing the bacteria from the blood. However, CRP was not completely protective at higher doses of S. pneumoniae. Since CRP provides complete protection against these doses in normal mice, the authors speculated that CRP's function is to slow the development of pneumococcal bacteremia until protective antibodies to capsular polysaccharide can be produced. C3 depletion decreased or abrogated the protective effects of CRP in xid mice, but not in normal mice.
Nakayama et al., J. Immunol., 132, 1336-40 1984) reports the results of injecting mice with 50-200 .mu.g of CRP and then immunizing them with type 3 S. pneumococci. The result was a diminished antibody response to the phosphorylcholine determinants on the bacteria which varied with the dose of CRP. However, antibodies were formed to other antigenic determinants on the S. pneumococci.
Hokama et al., J. Bacteriology, 83, 1017-1024 (1962) reports that carbonyl iron spherules, Diplococcus pneumoniae types IIs and XXVIIs and Serratia marcescens were phagocytosed more rapidly and in greater numbers by leukocytes of normal human blood after incubation with CRP. Similarly, Kindmark, Clin. Exp. Immunol., 8, 941-48 (1971) reports that CRP stimulated phagocytosis of Diplococcus pneumoniae, Staphylococcus aureus, Escherichia coli and Klebsiella aerogenes.
Gupta et al., J. Immunol., 137, 2173-79 (1986) teaches that CRP has been detected in immune complexes isolated from the sera of patients with acute rheumatic fever. Rheumatic fever is an acute inflammatory disease that may follow group A streptococcal pharyngitis. The other components of the immune complexes included streptolysin O and antibodies to streptolysin O.
However, Ballou et al., J. Lab. Clin. Med., 115, 332-38 (1990) teaches that highly purified CRP does not bind to immunoglobulin (monomeric or aggregated) or immune complexes. The article suggests that the reported presence of CRP in immune complexes may result from, or be facilitated by, an association of CRP with components of the immune complexes other than immunoglobulin, such as antigens or complement components.
Kilpatrick and Volanakis, J. Immunol., 134, 3364-70 (1985) reports that there is a CRP receptor on stimulated polymorphonuclear leukocytes (PMN). The authors also disclose that the ingestion of erythrocytes coated with pneumococcal C-polysaccharide and CRP by activated PMN is greater than ingestion of erythrocytes coated only with pneumococcal C-polysaccharide. Finally, the authors propose that CRP's function relates to its ability to specifically recognize foreign pathogens and damaged or necrotic host cells and to initiate their elimination by 1) interacting with the complement system or 2) interacting with inducible phagocytic receptors on neutrophils.
James et al., Dissertation Abstracts International, 41/08-B, 2963 (1980) teaches that CRP binds to a subset of mononuclear leukocytes, including 40% of the phagocytic monocytes and 3% of lymphocytes. Binding was influenced by several factors, including the form of the CRP molecule (i.e., modification of the CRP was required, either by complexing to a ligand or by heating to 63.degree. C).
Tebo et al., J. Immunol., 144, 231-38 (1990) teaches the presence of a receptor for CRP on monocytes. The article further discloses that a membrane receptor for CRP has been reported on neutrophils.
Kempka et al., J. Immunol., 144, 1004-1009 (1990) discloses results which the authors interpret to mean that CRP is a galactose-specific binding protein which, when associated to the surface of liver macrophages, functions as a receptor mediating galactose-specific endocytosis of particulate ligands.
CRP is a pentamer which consists of five identical subunits. The pentameric form of CRP is sometimes referred to as "native CRP." In about 1983, another form of CRP was discovered which is referred to as "modified-CRP" or "mCRP". mCRP has significantly different charge, size, solubility and antigenicity characteristics as compared to native CRP. Potempa et al., Mol. Immunol., 20, 1165-75 (1983). In particular, mCRP has a pI of 5.4, alpha globulin electrophoretic mobility and a molecular weight of about 22,000 in contrast to native CRP which has a pI of 6.4, gamma globulin electrophoetic mobility and a molecular weight of about 110,000. Id.; Potempa et al., Molec. Immunol., 24, 531-41 (1987). Also, mCRP has limited solubility and tends to aggregate in buffers of ionic strength 0.15, but remains soluble when the ionic strength is reduced to 0.015. Id. mCRP also differs from native CRP in binding characteristics; for instance, mCRP does not bind phosphorylcholine. Potempa et al., Molec. Immunol., 20, 1165-75 (1983); Chudwin et al., J. Allergy Clin. Immunol., 77, 216a (1986). Finally, mCRP differs from native CRP in its biological activity. See Potempa et al., Protides Biol. Fluids, 34, 287-290 (1986); Potempa et al., Inflammation, 12, 391-405 (1988).
The distinctive antigenicity of mCRP has been referred to as "neo-CRP." Neo-CRP antigenicity is expressed on:
1) CRP treated with acid, urea or heat under certain conditions (described below);
2) the primary translation product of DNA coding for CRP (preCRP); and
3) CRP immobilized on plastic surfaces. Potempa et al., Mol. Immunol., 20, 1165-75 (1983); Mantzouranis et al., Ped. Res., 18, 260a (1984); Samols et al., Biochem. J., 227, 759-65 (1985); Chudwin et al., J. Allergy Clin. Immunol., 77, 216a (1986); Potempa et al., Inflammation, 12, 391-405 (1988).
A molecule reactive with polyclonal antibody specific for neo-CRP has been identified on the surface of 10-25% of peripheral blood lymphocytes (predominantly NK and B cells), 80% of monocytes and 60% of neutrophils, and at sites of tissue injury. Potempa et al., FASEB J., 2, 731a (1988); Bray et al., Clin. Immunol. Newsletter, 8, 137-140 (1987); Rees et al., Fed. Proc., 45, 263a (1986). In addition, it has been reported that mCRP can influence the development of monocyte cytotoxicity, improve the accessory cell function of monocytes, potentiate aggregated-IgG-induced phagocytic cell oxidative metabolism, and increase the production of interleukin-1, prostaglandin E and lipoxygenase products by monocytes. Potempa et al., Protides Biol. Fluids, 34, 287-290 (1987); Potempa et al., Inflammation, 12, 391-405 (1988); Chu et al., Proc. Amer. Acad. Cancer Res., 28, 344a (1987); Potempa et al., Proc. Amer. Acad. Cancer Res., 28, 344a (1987); Zeller et al., Fed. Proc., 46, 1033a (1987); Chu et al., Proc. Amer. Acad. Cancer Res., 29, 371a (1988).
Chudwin et al., J. Allergy Clin. Immunol., 77, 216a (1986) teaches that mCRP can have a protective effect in mice challenged with gram-positive type 7F S. pneumoniae. Mice were injected intravenously with saline, native CRP, or mCRP. Thirty minutes later the mice received a lethal dose of S. pneumoniae. Survival at 10 days was as follows: 2/18 mice pretreated with saline; 7/12 mice pretreated with 200 .mu.g of native CRP; 12/18 mice pretreated with 10 .mu.g mCRP; and 5/6 mice pretreated with 100 .mu.g of mCRP. The authors speculate that CRP may be protective against bacterial infections by mechanisms other than phosphorylcholine binding and that CRP may have a wider role in bacterial host defenses than previously suspected through mCRP (which does not bind phosphorylcholine).
To Applicant's knowledge, there have been no reports that mCRP is protective against any other kind of bacterial infection.
For a brief review of CRP and mCRP, see Gotschlich, Ann. N.Y. Acad. Sci., 557, 9-18 (1989). Kilpatrick and Volanakis, Immunol. Res., 10, 43-53 (1991) provides a recent review of CRP.
Finally, Applicant wishes to draw attention to certain co-pending applications on which he is named as a co-inventor. U.S. application Ser. No. 07/582,884, filed Oct. 3, 1990, relates to the use of mCRP to bind immune complexes. This application was filed as a national application of PCT application US89/01247 (published as WO 89/09628 on Oct. 19, 1989) and is a continuation-in-part of U.S. application 07/176,923, filed Apr. 4, 1988, now abandoned. Applicant is also named as a co-inventor on U.S. application Ser. No. 07/374,166, filed Jun. 29, 1989, a continuation-in-part of application Ser. No., 07/372,442 filed Jun. 27, 1989, now abandoned. This application describes and claims monoclonal antibodies selectively reactive with epitopes found on native CRP, mCRP or both Finally, being filed on even date herewith is an application entitled "Method Of Treating Viral Infections" which relates to the use of mCRP to treat such infections.